A. Innate and Adaptive Immunity
Autoimmune diseases, are currently clinically defined by (i) humoral or autoantibody response to a self antigen, e.g. Graves' primary hyperthyroidism with antibodies to the TSH receptor, or (ii) cellular response wherein immune cells destroy nonimmune cells from which the self-antigen is derived, e.g. the thyrocyte (Hashimoto's thyroiditis) or pancreatic β-islet cell (Type 1 diabetes) (I. Roitt, Essential Immunology, 7th ed., 312-346 (1991)). Many autoimmune diseases are in fact a combination of both phenomena (I. Roitt, Essential Immunology, 7th ed., 312-346 (1991)); thus, Hashimoto's and Type 1 diabetes also have auto-antibodies, anti-thyroid peroxidase (TPO) or anti-glutamic acid decarboxylase (GAD)/Islet Cell. Additionally, autoimmune diseases often have a significant inflammatory component including increases in adhesion molecules, e.g. vascular cell adhesion molecule-1 (VCAM-1), and altered leukocyte adhesion to the vasculature, e.g., colitis, systemic lupus, systemic sclerosis, and the vascular complications of diabetes (I. Roitt, Essential Immunology, 7th ed., 312-346 (1991); S. A. Jimenez, et al., Ann Intern Med, 140:37-50 (2004)).
Recent studies demonstrate a formidable link between the Toll-like receptor (TLR) signaling pathway of innate immunity and the slower, more deliberate adaptive immune system that characterizes humoral and cellular autoimmunity (K. S. Michelsen, et al., Proc Natl Acad Sci USA, 101:10679-84 (2004); G. Pasterkamp, et al., Eur J Clin Invest, 34:328-34 (2004); K. Takeda, et al., Annu Rev Immunol, 21:335-76 (2003); K. Takeda, et al., Cell Microbiol, 5:143-53 (2003); R. J. Ulevitch, J Infect Dis, 187 Suppl 2:S351-5 (2003); L. Steinman, Science, 305:212-6 (2004); L. D. Kohn, et al., Research Ohio, In press, (2005); N. Harii, et al., Mol Endocrinol, 19:1231-50 (2005); D. Devendra, et al., Clin Immunol, 111:225-33 (2004); L. Wen, et al., J Immunol, 172:3173-80 (2004); H. Oshiumi, et al., Nat Immunol, 4:161-7 (2003); M. Yamamoto, et al., J Immunol, 169:6668-72 (2002); M. Miettinen, et al., Genes Immun, 2:349-55 (2001); L. Alexopoulou, et al., Nature, 413:732-8 (2001); G. Andonegui, et al., J Clin Invest, 111:1011-1020 (2003); C. Fiocchi, Gastroenterology, 115:182-205 (1998); E. Cario, et al., Infect Immun, 68:7010-7 (2000)). Innate immunity is a protective immune cell response that functions rapidly to fight environmental insults including, but not limited to, bacterial or viral agents. Adaptive immunity is a slower response, which involves differentiation and activation of naive T lymphocytes into T helper 1 (Th1) or T helper 2 (Th2) cell types (I. Roitt, Essential Immunology, 7th ed., 312-346, (1991)). Th1 cells mainly promote cellular immunity, whereas Th2 cells mainly promote humoral immunity. Though primarily a host protective system, pathologic expression of the innate immunity signals emanating from the TLR pathway are now implicated in initiating autoimmune-inflammatory diseases.
Therapies for autoimmune-inflammatory endocrine or non-endocrine diseases are largely aimed at treating the symptoms of the disease. For the most part, the underlying genetic susceptibilities are poorly defined, are multiple, are often not disease specific, and are largely not readily amenable to therapy. Immunosuppressive agents that are used to treat autoimmune-inflammatory diseases largely target the immune cell response or the cytokines they produce. They are only partially effective in inducing remission (methimazole in Graves'), toxic (cyclosporin for Type 1 diabetes), or simply palliative (anti-inflammatory corticosteroids for colitis or systemic lupus). The involvement of TLR in autoimmune-inflammatory diseases raises the possibility that diagnosis and treatment must undergo a re-alignment (K. S. Michelsen, et al., Proc Natl Acad Sci USA, 101: 10679-84 (2004); G. Pasterkamp, et al., Eur J Clin Invest, 34:328-34 (2004); L. D. Kohn, et al., Research Ohio, In press, (2005); N. Harii, et al., Mol Endocrinol, 19:1231-50 (2005); D. Devendra, et al., Clin Immunol, 111:225-33 (2004); L. Wen, et al., J Immunol, 172:3173-80 (2004); H. Oshiumi, et al., Nat Immunol, 4:161-7 (2003); M. Yamamoto, et al., J Immunol, 169:6668-72 (2002); M. Miettinen, et al., Genes Immun, 2:349-55 (2001); L. Alexopoulou, et al., Nature, 413:732-8 (2001); G. Andonegui, et al., J Clin Invest, 111:1011-1020 (2003); C. Fiocchi, Gastroenterology, 115:182-205 (1998); E. Cario, et al., Infect Immun, 68:7010-7 (2000)).
Thus, despite our knowledge that many autoimmune-inflammatory diseases were induced or worsened by an environmental agent, e.g. smoking or viral infections, little was known of the details by which this induction-signal process worked, nor was there a therapy to block this induction-signal process (I. Roitt, Essential Immunology, 7th ed., 312-346, (1991); J. George, et al., Scand J Immunol, 45:1-6 (1997); C. Nagata, et al., Int J Dermatol, 34:333-7 (1995)).
Thus, the recent description of TLR and the TLR signal mechanism of innate immunity, upon which adaptive (humoral or cell-mediated) immunity depends has created an opportunity to develop of a new class of drugs as well as new diagnostic paradigms (L. D. Kohn, et al., Research Ohio, In press, (2005); N. Harii, et al., Mol Endocrinol, 19:1231-50 (2005); D. Devendra, et al., Clin Immunol, 111:225-33 (2004); L. D. Kohn, et al., U.S. patent application Ser. No. 10/801,986 (2004); L. D. Kohn, et al., U.S. patent application Ser. No. 10/912,948 (2004)).
By attacking the innate immune induction event of autoimmune/inflammatory disease, early identification of the induction signal event or environmental insult in a person at risk and initiation of therapy post induction or during the latency period of disease onset could allow therapy to be more effective, prevent or retard cell destruction, avoid long term inflammatory complications, enhance quality of life, and decrease associated medical costs. Since, there is increasing evidence that the atherosclerotic process and cardiovascular disease, i.e. the vascular complications of type 2 and type 1 diabetes, exhibit similar mechanisms involving TLR and a pathologic innate immune response, they too can benefit from the same treatment paradigm, despite being currently considered late stage phenomena (K. S. Michelsen, et al., Proc Natl Acad Sci USA, 101:10679-84 (2004); G. Pasterkamp, et al., Eur J Clin Invest, 34:328-34 (2004); H. M. Dansky, et al., Arterioscler Thromb Vasc Biol, 21:1662-7 (2001); P. E. Szmitko, et al., Circulation, 108:2041-8 (2003); P. E. Szmitko, et al., Circulation, 108:1917-23 (2003); M. I. Cybulsky, et al., Can J Cardiol, 20 Suppl B:24B-8B (2004); P. M. Ridker, et al., Circulation, 109:IV6-19, (2004)). No such method exists although it is considered important.
B. Toll-Like Receptors and Signaling
At the end of the 20th century, Toll-like receptors (TLRs) were shown to be essential to induce expression of genes involved in inflammatory responses. Since their description, there has been rapid progress in our understanding that TLRs and the innate immune system is a critical step in the development of antigen-specific acquired immunity. This is recently reviewed by several groups (K. Takeda, et al., Int Immunol, 17:1-14 (2005); B. Beutler, Nature, 430:257-63 (2004); K. S. Michelsen, et al., J Immunol, 173:5901-7 (2004)); the material following is largely derived from one (K. Takeda, et al., Int Immunol, 17:1-14 (2005)) but is common to all (B. Beutler, Nature, 430:257-63 (2004); K. S. Michelsen, et al., J Immunol, 173:5901-7 (2004)) and represents only the current thoughts in a rapidly developing area.
The TLR Family. Mammalian TLRs comprise a large family consisting of at least 10 functional members such as TLR1-9 which are conserved between the human and mouse. The cytoplasmic portion of TLRs shows high similarity to that of the IL-1 receptor family and is termed a Toll/IL-1 receptor (TIR) domain. Despite this similarity, the extracellular portions of TLR are structurally unrelated. The IL-1 receptors possess an immunoglobulin-like domain, whereas TLRs bear leucine-rich repeats (LRRs) in the extracellular domain. TLRs play important roles in recognizing specific signature molecules derived from pathogens including bacteria, fungi, protozoa and viruses, derived from their invasion of cells, or resultant from the effects of noxious environmental stimuli which cause cell damage.
Toll-like Receptors 1, 2, and 6 (TLR1, TLR2 and TLR6). TLR2 recognizes a variety of lipoproteins/lipopeptides from various pathogens, e.g. Gram-positive bacteria, mycobacteria, Trypanosoma cruzi, fungi and Treponema (K. Takeda, et al., Annu Rev Immunol, 21:335-76 (2003)). In addition, TLR2 reportedly recognizes LPS preparations from non-enterobacteria such as Leptospira interrogans, Porphyromonas gingivalis and Helicobacter pylori. These LPS structurally differ from the typical LPS of Gram-negative bacteria recognized by TLR4 in the number of acyl chains in the lipid A component, which presumably confers differential recognition; thus, LPS from P. gingivalis only poorly activates TLR4 (M. Hashimoto, et al., Int Immunol, 16:1431-7 (2004)).
There are two proposed explanations that could explain why TLR2 recognizes a wide spectrum of microbial components. The first is that TLR2 forms heterophilic dimers with other TLRs such as TLR1 and TLR6, both of which are structurally related to TLR2. The second involves interactions (B. N. Gantner, et al., J Exp Med, 197:1107-17 (2003)) with distinct types of receptors such as dectin-1, a lectin family receptor for the fungal cell wall component beta-glucan. Thus, TLR2 recognizes a wide range of microbial products through functional cooperation with several proteins that are either structurally related or unrelated to TLR.
Toll-like receptor 3 (TLR3). Expression of human TLR3 in non-responsive cells confers enhanced activation of NF-κB in response to dsRNA. In addition, TLR3-deficient mice are impaired in their response to dsRNA (L. Alexopoulou, et al., Nature, 413:732-8 (2001)) which is produced by most viruses during their replication and which induces the synthesis of type I interferons (IFN-α/β). Type I IFNs induce anti-viral and immunostimulatory activities in the cells. Thus, TLR3 is implicated in the recognition of dsRNA and viruses and the antiviral gene response thereto.
Toll-like Receptor 4 (TLR4). TLR4 is an essential receptor for LPS recognition (A. Poltorak, et al., Science, 282:2085-8 (1998); K. Hoshino, et al., J Immunol, 162:3749-52 (1999)). In addition, TLR4 is implicated in the recognition of endogenous ligands, such as heat shock proteins (HSP60 and HSP70), domain A of fibronectins, as well as oligosaccharides of hyaluronic acid, heparan sulfate and fibrinogen. However, since these endogenous ligands require very high concentrations to activate TLR4, contamination by LPS is suspected.
Toll-like Receptor 5 (TLR5). Expression of human TLR5 in CHO cells confers response to flagellin, a monomeric constituent of bacterial flagella (F. Hayashi, et al., Nature, 410:1099-103 (2001)). TLR5 is expressed on the basolateral side of intestinal epithelial cells and intestinal endothelial cells of the subepithelial compartment. Further, flagellin activates lung epithelial cells to induce inflammatory cytokine production and a stop codon polymorphism in TLR5 has been associated with susceptibility to pneumonia caused by the flagellated bacterium Legionella pneumophila. These findings indicate the important role of TLR5 in microbial recognition at the mucosal surface of mammalian cells.
Toll-like Receptors 7 and 8 (TLR7 and TLR8). TLR7 and TLR8 are structurally highly conserved proteins, which recognize guanosine- or uridine-rich, single-stranded RNA (ssRNA) from viruses such as human immunodeficiency virus, vesicular stomatitis virus and influenza virus (F. Heil, et al., Science, 303:1526-9 (2004); S. S. Diebold, et al., Science, 303:1529-31 (2004); J. M. Lund, et al., Proc Natl Acad Sci USA, 101:5598-603 (2004)). ssRNA is abundant in the host, but usually the host-derived ssRNA is not detected by TLR7 or TLR8. This might be due to the fact that TLR7 and TLR8 are expressed in the endosome, and host-derived ssRNA is not delivered to the endosome (see below).
Toll-like Receptor 9 (TLR9). TLR9 is a receptor for CpG DNA (H. Hemmi, et al., Nature, 408:740-5 (2000)). Bacterial and viral DNA contains unmethylated CpG motifs, which confer its immunostimulatory activity. In vertebrates, the frequency of CpG motifs is severely reduced and the cytosine residues of CpG motifs are highly methylated, leading to abrogation of the immunostimulatory activity. Structurally, there are at least two types of CpG DNA: B/K-type CpG DNA is a potent inducer of inflammatory cytokines such as IL-12 and TNF-α; A/D-type CpG DNA has a greater ability to induce IFN-α production from plasmacytoid dendritic cells (PDC), In addition to recognizing bacterial and viral CpG DNA, TLR9 is involved in pathogenesis of autoimmune disorders. Thus it may be important in Graves' autoimmune hyperthyroidism and mediates production of rheumatoid factor by auto-reactive B cells (G. A. Viglianti, et al., Immunity, 19:837-47 (2003)). Similarly, internalization by the Fc receptor can cause TLR9 mediated PDC induction of IFN-α by immune complexes containing IgG and chromatin, which are implicated in the pathogenesis of systemic lupus erythematosus (SLE) (M. W. Boule, et al., J Exp Med, 199:1631-40 (2004)). Thus, TLR9 appears to be involved in the pathogenesis of several autoimmune diseases through recognition of the chromatin structure. Chloroquine, which is clinically used for treatment of rheumatoid arthritis and SLE, is currently presumed to block TLR9-dependent signaling through inhibition of the pH-dependent maturation of endosomes by neutralizing acidification in the vesicle (H. Hacker, et al., Embo J, 17:6230-40 (1998)).
Toll-like Receptor 11 (TLR11). The most recently identified TLR11 has been shown to be expressed in bladder epithelial cells and mediate resistance to infection by uropathogenic bacteria in mouse (D. Zhang, et al., Science, 303:1522-6 (2004)).
Subcellular Localization of Some TLRs. Individual TLRs are differentially distributed within the cell. TLR1, TLR2, TLR3 and TLR4 are expressed on the cell surface; in contrast, TLR3, TLR7, TLR8 and TLR9 have been shown to be expressed in intracellular compartments such as endosomes. TLR3-, TLR7- or TLR9-mediated recognition of their ligands has been shown to require endosomal maturation and processing. Thus, for example, TLR9 is recruited from the endoplasmic reticulum upon non-specific uptake of CpG DNA (H. Hacker, et al., Embo J, 17:6230-40 (1998); E. Latz, et al., Nat Immunol, 5:190-8 (2004); C. A. Leifer, et al., J Immunol, 173:1179-83 (2004)). When either nonimmune cells that become antigen presenting cells, macrophages, monocytes, or dendritic cells engulf bacteria by phagocytosis, they degrade the bacteria and release CpG DNA in phagosomes-lysosomes or in endosomes-lysosomes where they can interact TLR9.
Similarly, as another example, when viruses invade cells by receptor-mediated endocytosis, the viral contents are exposed to the cytoplasm by fusion of the viral membrane with the endosomal membrane. This results in exposure of TLR ligands such as dsRNA, ssRNA and CpG DNA to TLR9 in the phagosomal/lysosomal or endosomal/lysosomal compartments.
TLR-independent Recognition of Micro-organisms—dsRNA Transfection De Novo or RNA/DNA Introduction By viruses—Can Nevertheless Activate TLR Signaling Pathways. Although TLR3 is involved in the recognition of viral-derived dsRNA, the impairment observed in TLR3-deficient mice is only partial (L. Alexopoulou, et al., Nature, 413:732-8 (2001); M. Yamamoto, et al., Science, 301:640-3 (2003)). Thus, introduction of dsRNA into the cytoplasm of dendritic cells leads to the induction of type I IFNs independent of TLR3 (S. S. Diebold, et al., Nature, 424:324-8 (2003)). Although PKR is implicated in dsRNA recognition, it is still controversial if it plays a critical role in dsRNA-induced type I IFN expression (E. J. Smith, et al., J Biol Chem, 276:8951-7 (2001)).
Recently, one key molecule that mediates TLR3-independent dsRNA recognition was shown to be Retinoic acid-inducible gene I (RIG-I). RIG-1 encodes a DExD/H box RNA helicase containing a caspase recruitment domain that augments dsRNA-dependent activation of the IRF-3-dependent promoter.
The nucleotide-binding oligomerization domain (NOD) family of proteins also plays an important role in the TLR-independent recognition of intracellular bacteria.
NOD1 contains a caspase-recruitment domain (CARD), a NOD domain and a C-terminal LRR domain. Overexpression of NOD1 enables cells to respond to peptidoglycans (PGN) which are recognized by TLR2 (O. Takeuchi, et al., Immunity, 11:443-51, (1999)); c-D-glutamyl-meso diaminopimelic acid (iE-DAP) is the minimal PGN structure required. NOD2 shows structural similarity to NOD1, but possesses two CARD domains and the essential structure recognized by NOD2 is a muramyl dipeptide MurNAc-L-Ala-D-isoGln (MDP) derived from PGN. MDP is found in almost all bacteria, whereas iE-DAP is restricted to Gram-negative bacteria.
Mutations in the NOD2 gene have been shown to be associated with Crohn's disease (Y. Ogura, et al., Nature, 411:603-6 (2001); J. P. Hugot, et al., Nature, 411:599-603 (2001)), result in enhanced NF-κB activation and may contribute to enhanced NF-κB activity and Th1 responses in Crohn's disease patients (T. Watanabe, et al., Nat Immunol, 5:800-8 (2004)). NOD2 mutations also lead to an increase in NF-κB activity and are associated with Blau syndrome, a disease characterized by granulomatous arthritis, uveitis and skin rash (C. Miceli-Richard, et al., Nat Genet, 29:19-20 (2001)).
Rip2/RICK, a serine/threonine kinase, has a CARD domain in its C-terminal portion and an N-terminal catalytic domain that shares sequence similarity with Rip, a factor essential for NF-κB activation through the TNF receptor. NODs and Rip2/RICK interact via their respective CARD domains, and induce recruitment of the IKK complex to the central region of Rip2/RICK. This in turn leads to activation of NF-κB.
TLR Signaling Pathways—MyD88 Pathway and NF-κB/MAP Kinase Signals. In the signaling pathways downstream of the TIR domain, a TIR domain-containing adaptor, MyD88, was the first shown to be essential for induction of inflammatory cytokines such as TNF-α and IL-12 through all TLRs (F. Hayashi, et al., Nature, 410:1099-103 (2001); H. Hemmi, et al., Nat Immunol, 3:196-200 (2002); O. Takeuchi, et al., Int Immunol, 12:113-7, (2000); T. Kawai, et al., Immunity 11:115-22, (1999); M. Schnare, et al., Curr Biol, 10:1139-42 (2000); H. Hacker, et al., J Exp Med, 192:595-600 (2000)). However, activation of specific TLRs led to slightly different patterns of gene expression profiles. For example, activation of TLR3 and TLR4 signaling pathways resulted in induction of type I interferons (IFNs), but activation of TLR2- and TLR5-mediated pathways did not (V. Toshchakov, et al., J Endotoxin Res, 9:169-75 (2003); K. Hoshino, et al., Int Immunol, 14:1225-31 (2002); S. Doyle, et al., Immunity, 17:251-63 (2002)). TLR7, TLR8 and TLR9 signaling pathways also lead to induction of Type I IFNs through mechanisms distinct from TLR3/4-mediated induction (H. Hemmi, et al., J Immunol, 170:3059-64 (2003); T. Ito, et al., J Exp Med, 195:1507-12 (2002)). Thus, individual TLR signaling pathways are divergent, although MyD88 is common to all TLRs. It has thus become clear that there are MyD88-dependent and MyD88-independent pathways.
The MyD88-dependent pathway is analogous to signaling by the IL-1 receptors. As currently perceived, MyD88, harboring a C-terminal TIR domain and an N-terminal death domain, associates with the TIR domain of TLRs. Upon stimulation, MyD88 recruits IRAK-4 to TLRs through interaction of the death domains of both molecules, and facilitates IRAK-4-mediated phosphorylation of IRAK-1. Activated IRAK-1 then associates with TRAF6, leading to the activation of two distinct signaling pathways. One pathway leads to activation of AP-1 transcription factors through activation of MAP kinases. Another pathway activates the TAK1/TAB complex, which enhances activity of the IκB kinase (IKK) complex. Once activated, the IKK complex induces phosphorylation and subsequent degradation of IκB, which leads to nuclear translocation of transcription factor NF-κB. The MyD88-dependent pathway plays a crucial role and is essential for inflammatory cytokine production through all TLRs. Thus, MyD88-deficient mice do not show production of inflammatory cytokines such as TNF-α and IL-12p40 in response to all TLR ligands (F. Hayashi, et al., Nature, 410:1099-103, (2001); H. Hemmi, et al., Nat Immunol, 3:196-200 (2002); O. Takeuchi, et al., Int Immunol, 12:113-7 (2000); T. Kawai, et al., Immunity, 11:115-22 (1999); M. Schnare, et al., Curr Biol, 10:1139-42 (2000); H. Hacker, et al., J Exp Med, 192:595-600 (2000)).
A MyD88 related TIR domain-containing molecule: TIRAP (TIR domain-containing adaptor protein)/Mal (MyD88-adaptor-like) (T. Horng, et al., Nat Immunol, 2:835-41 (2001); K. A. Fitzgerald, et al., Nature, 413:78-83 (2001)) has been shown to be essential for the MyD88-dependent signaling pathway via TLR2 and TLR4. Thus, TIRAP/Mal-deficient macrophages show impaired inflammatory cytokine production in response to TLR4 and TLR2 ligands (T. Horng, et al., Nature, 420:329-33 (2002); M. Yamamoto, et al., Nature, 420:324-9 (2002)) but are not impaired in their response to TLR3, TLR5, TLR7 and TLR9 ligands.
MyD88-independent/TRIF-dependent Pathway and IRF-3/Type 1 IFN Signaling. TLR4 ligand-induced production of inflammatory cytokines is not observed in MyD88-deficient macrophages, however activation of NF-κB is observed with delayed kinetics (T. Kawai, et al., J Immunol, 167:5887-94 (2001)). Thus, a MyD88-independent component exists.
In TLR3- and TLR4-mediated signaling pathways, activation of IRF-3 and induction of IFN-β are observed in a MyD88-independent manner. The TIR domain-containing adaptor, TRIF, is essential for the MyD88-independent pathway; however, in the case of TLR4, but not TLR3, the TIR domain-containing adaptor, TRAM, is also involved in the TRIF-dependent activation of IRF-3 and induction of IFN-β- and IFN-inducible genes pathway as evidenced in TRAM-deficient mice or by RNAi-mediated knockdown (K. A. Fitzgerald, et al., J Exp Med, 198:1043-55 (2003); M. Yamamoto, et al., Nat Immunol, 4:1144-50 (2003); H. Oshiumi, et al., J Biol Chem, 278:49751-62 (2003)).
Non-typical IKKs, IKKi/IKKe and TBK1, mediate activation of IRF-3 downstream of TRIF as well as the late phase of NF-κB activation in a MyD88-independent manner (T. Kawai, et al., J Immunol, 167:5887-94 (2001)). Activation of IRF-3 leads to production of IFN-β. IFN-β in turn activates Stat1 and induces several IFN-inducible genes (V. Toshchakov, et al., J Endotoxin Res, 9:169-75 (2003); K. Hoshino, et al., Int Immunol, 14:1225-31 (2002); S. Doyle, et al., Immunit, 17:251-63, (2002)). The physiological role of TRIF was demonstrated by generation of TRIF-deficient or TRIF-mutant mice which showed no activation of IRF-3 and had impaired expression of IFN-β- and IFN-inducible genes in response to TLR3 and TLR4 ligands (S. S. Diebold, et al., Nature, 424:324-8 (2003)).
In TRIF- and TRAM-deficient mice, inflammatory cytokine production induced by TLR2, TLR7 and TLR9 ligands was observed, as well as TLR4 ligand-induced phosphorylation of IRAK-1 (S. S. Diebold, et al., Nature, 424:324-8 (2003); M. Yamamoto, et al., Nat Immunol, 4:1144-50 (2003)). These findings indicate that the MyD88-dependent pathway is not impaired in these mice. However, TLR4 ligand-induced inflammatory cytokine production was also not observed in TRIF- and TRAM-deficient mice. Therefore, activation of both the MyD88-dependent and MyD88-independent/TRIF-dependent components is believed to be required for the TLR3/4-induced inflammatory cytokine production.
Key molecules that mediate IRF-3 activation have been revealed to be non-canonical IKKs, TBK1 and IKKi/IKKe. Thus, introduction of TBK1 or IKKi/IKKe, but not IKKb, resulted in phosphorylation and nuclear translocation of IRF-3. Also, RNAi-mediated inhibition of TBK1 or IKKi/IKKe expression led to impaired induction of IFN-β in response to viruses and dsRNA (S. Sharma, et al., Science, 300:1148-51, (2003)).
The Mechanisms of MyD88-independent TLR Signaling of Both IRF-3 and NF-κB Pathways by TLR3: The TIR domain of TRIF is located in the middle portion of this molecule, flanked by the N-terminal and C-terminal portions. Both N-terminal and C-terminal portions of TRIF mediate activation of the NF-κB-dependent promoter, whereas only the N-terminal portion is involved in IFN-β promoter activation (M. Yamamoto, et al., J Immunol, 169:6668-72 (2002)). Accordingly, the N-terminal portion of TRIF was shown to associate with IKKi/IKKe and TBK1, which mediate IRF-3-dependent IFN-β induction (K. A. Fitzgerald, et al., Nat Immunol, 4:491-6 (2003); S. Sato, et al., J Immunol, 171:4304-10 (2003)). The N-terminal portion of TRIF was also shown to associate with TRAF6 (S. Sato, et al., J Immunol, 171:4304-10 (2003); Z. Jiang, et al., Proc Natl Acad Sci USA, 101:3533-8 (2004)); TRAF6 is critically involved in TLR-mediated NF-κB activation (J. Gohda, et al., J Immunol, 173:2913-7 (2004)), The C-terminal portion of TRIF was shown to associate with RIPI (E. Meylan, et al., Nat Immunol, 5:503-7 (2004)); thus, RIPI was shown to be responsible for NF-κB activation that originates from the C-terminal portion of TRIF.
Negative Regulation of TLR Signaling. Stimulation of TLRs by microbial components triggers the induction of inflammatory cytokines such as TNF-α, IL-6 and IL-12. When all these cytokines are produced in excess, they induce serious systemic disorders with a high mortality rate in the host. It is therefore not surprising that organisms have evolved mechanisms for modulating their TLR-mediated responses. TLR signaling pathways are negatively regulated by several molecules. IRAK-M inhibits dissociation of IRAK-1/IRAK-4 complex from the receptor. MyD88s blocks association of IRAK-4 with MyD88. SOCS1 is likely to associate with IRAK-1 and inhibits its activity. TRIAD3A induces ubiquitination-mediated degradation of TLR4 and TLR9. TIR domain-containing receptors SIGIRR and T1/ST2 are also shown to negatively modulate TLR signaling. Thus, several molecules are postulated to negatively modulate TLR signaling pathways and in combination may normally finely coordinate the TLR signaling pathway to limit exaggerated innate responses causing harmful disorders.
Exposure to microbial components such as LPS results in a severely reduced response to a subsequent challenge by LPS, termed endotoxin or LPS tolerance. Several negative regulation mechanisms are also shown to be involved in LPS tolerance (H. Fan, et al., J Endotoxin Res, 10:71-84 (2004)).
C. IRF-1 Signaling Induced by Overexpressed TLR3 or TLR4 Signaling is Critical in Autoimmune Inflammatory Disease
The regulatory effect of IRF-1 has been reported in several in vitro and in vivo models of autoimmune-inflammatory diseases: Arthritis (A. Shiraishi, et al., J Immunol, 159:3549-54 (1997); T. Inoue, et al., J Rheumatol, 28:1229-37 (2001); S. John, et al., J Rheumatol, 28:1752-5 (2001)), colitis (M. Clavell, et al., J Pediatr Gastroenterol Nutr, 30:43-7 (2000)); (M. Kennedy, et al., Int J Mol Med, 4:437-43 (1999)), neurological inflammation (M. Delgado, et al., J Immunol, 162:4685-96 (1999); U. Schlomann, et al., J Neurosci, 20:7964-71 (2000)), cerebral ischemia (C. Iadecola, et al., J Exp Med, 189:719-27 (1999); W. Paschen, et al., Neuroreport, 9:3147-51 (1998)); V. L. Raghavendra Rao, et al., J Neurochem, 83:1072-86 (2002)), lung injury (V. R. Sunil, et al., Am J Physiol Lung Cell Mol Physiol, 282:L872-80 (2002)), myositis (S. Matsubara, et al., J Neuroimmunol, 119:223-30 (2001)), myocarditis (K. Azzam-Smoak, et al., Virology, 298:20-9 (2002); S. Kawamoto, et al., J Virol, 77:9622-31 (2003); R. Kamijo, et al., Science, 263:1612-5 (1994); J. R. Allport, et al., J Exp Med, 186:517-527 (1997)), endotoxic shock (G. Andonegui, et al., J Clin Invest, 111:1011-1020 (2003); V. L. Raghavendra Rao, et al., J Neurochem, 83:1072-86 (2002); S. Heinz, et al., J Biol Chem, 278:21502-9 (2003); C. W. Wieland, et al., Infect Immun, 70:1352-8 (2002); Y. Pang, et al., Brain Res, 914:15-22 (2001); O. Kobayashi, et al., Am J Physiol Gastrointest Liver Physiol, 281:688-96, (2001)), diabetes (A. Akabane, et al., Biochem Biophys Res Commun, 215:524-30 (1995); M. S. Baker, et al., Surgery, 134:134-41 (2003); C. A. Gysemans, et al., Diabetologia, 44:567-74 (2001); A. E. Karlsen, et al., J Clin Endocrinol Metab, 85:830-6 (2000); T. Nakazawa, et al., J Autoimmun 17:119-25, (2001)), hepatitis (B. Jaruga, et al., Am J Physiol Gastrointest Liver Physiol, 287:G1044-52 (2004); P. M. Pitha, et al., Biochimie, 80:651-8 (1998)), systemic lupus erythematosus (SLE), (K. M. Pollard, et al., Ann N Y Acad Sci, 987:236-9 (2003)), and a multifocal inflammatory model with autoimmune components (N. L. Mccartney-Francis, et al., J Immunol, 169:5941-7 (2002)). IRF-1 is implicated in patients with, autoimmune myocarditis associated with viral infection in human and in rodent models (K. Bachmaier, et al., Circulation, 96:585-91 (1997)).
IRF-1 can up-regulate the inflammatory immune response at the innate and adaptive level by increasing the inflammatory gene expression in macrophages, dendritic cells and CD-4 T cells. Thus, upregulation of IRF-1 gene expression can increase the expression of inflammatory mediators such as arachidonic acid signaling, COX-1 and, COX-2 enzymes (X. Teng, et al., Am J Physiol Cell Physiol, 282:C144-52, (2002)), chemokines (M. S. Baker, et al., Surgery, 134:134-41 (2003); Y. Ohmori, et al., J Leukoc Biol, 69:598-604 (2001)), iNOS (M. Delgado, et al., J Immunol, 162:4685-96 (1999); M. S. Baker, et al., Surgery, 134:134-41 (2003); X. Teng, et al., Am J Physiol Cell Physiol, 282:C144-52 (2002); Y. Ohmori, et al., J Leukoc Biol, 69:598-604 (2001)), IL-12 p40 (M. Clavell, et al., J Pediatr Gastroenterol Nutr, 30:43-7 (2000); C. Feng, et al., Int Immunol, 11:1185-94 (1999)) Type 1 IFN-α and -β (L. A. Eader, et al., Cell Immunol, 157:211-22 (1994); S. Kirchhoff, et al., Eur J Biochem, 261:546-54 (1999)), as well as the pro-inflammatory cytokines TNF-α, IL1-β, IL-6, IL-12 and INF-γ. IRF-1 gene overexpression may thus induce autoimmune-inflammatory diseases by its effects on macrophages, dendritic cells and CD4±Th1 cell lymphocytic cells.
Despite information implicating the importance of IRF-1 signaling in macrophages, dendritic cells and CD4±Th1 cell lymphocytic cells, comparable effects, after TLR3 or TLR4 mediated increases of IRF-1 in nonimmune cells, have been less clear. However, studies of the effects of methimazole, methimazole derivatives, and tautomeric cyclic thiones, particularly phenylmethimazole (C10) related to Hashimoto's thyroiditis, Colitis, toxic shock, and atherosclerosis summarized herein establish the importance of its overexpression in nonimmune cells associated with or caused by TLR3 or TLR4 signal overexpression.
D. IRF-1 Signalling Induced by Overexpressed TLR4Signaling is Critical in Atherosclerosis
Leukocyte adhesion is central to atherosclerosis, an autoimmune-inflammatory disease. One of the earliest steps in the development of atherosclerotic lesions is the adhesion of leukocytes (monocytes and lymphocytes) to the apical surface of the endothelium and subsequent migration across the endothelium into the subendothelial space at select anatomical sites in the arterial tree. This process occurs through a cascade of adhesive events. This adhesion cascade is mediated, in part, by binding of molecules present on the surface of the leukocyte (e.g. β1 integrins) to adhesion molecules on the surface of the endothelium (e.g VCAM-1). Subsequent to migrating into the extravascular space, the monocyte-derived macrophages ingest lipids and become foam cells. Activation of the recruited leukocytes is believed to induce release of important mediators of inflammation (e.g. pro-inflammatory cytokines) that serve to continue the process of lesion development. Smooth muscle cells are recruited to the fatty spot and, together with the foam cells and lymphocytes, form the fatty streak (intermediate lesion). This entire process can continue leading to a fibrofatty lesion and ultimately to a fibrous plaque. Throughout plaque development, the vascular endothelium remains intact. Since the mechanisms of atherogenesis are similar to those present in “general” pathological inflammation, atherosclerosis is often considered a disease of pathological inflammation. Indeed, it has recently been shown that inhibition of the potent pro-inflammatory cytokine TNF-α reduces atherosclerosis in a murine model (L. Branen, et al., Arterioscler Thromb Vasc Biol, 24:2137-42 (2004)).
Endothelial cell adhesion molecules (ECAMs), which are known to participate in leukocyte recruitment during pathological inflammation, (e.g VCAM-1, E-selectin and ICAM-1), have been shown to be up-regulated at sites of inflammation and to contribute to disease progression and/or tissue damage by virtue of their role in leukocyte adhesion (F. W. Luscinskas, et al., Annu. Rev. Med., 47:413-421 (1996)). VCAM-1 has received the most interest in the context of atherosclerosis. VCAM-1 has been observed in a localized fashion on aortic endothelium that overlies early foam cell lesions (M. I. Cybulsky, et al., Science, 251:788-791 (1991)) and has been shown to play an important role in monocyte and lymphocyte adhesion to and migration across the endothelium (F. W. Luscinskas, et al., J. Cell Biol., 125:1417-27 (1994); C. L. Ramos, et al., Circ. Res., 84:1237-44 (1999)). Studies with the Apolipoprotein E-deficient (ApoE−/−) mouse, a well-accepted model of human atherosclerosis, revealed that VCAM-1 is present on endothelium at lesion-prone sites (as early as 5 weeks) and developed lesions (Y. Nakashima, et al., Arterioscler. Thromb. Vasc. Biol., 18:842-51 (1998)). Monocytes exhibit greatly increased adhesion to carotid arteries isolated from ApoE−/− mice compared to carotid arteries isolated from wild-type mice and this increased adhesion is mediated, in part, by VCAM-1 (C. L. Ramos, et al., Circ. Res., 84:1237-44 (1999)).
The expression of ECAMs is regulated, in part, by pro-inflammatory cytokines (e.g. TNF-α) which increase the activity of certain transcription factors (e.g. NF-κB) (M. J. May, et al., Immunol. Today, 19:80-88 (1998)) and IRF-1 (A. S, Neish, et al., Mol. Cell. Biol., 15:2558-2569 (1995)). The activated or increased transcription factors bind to promoter, elements on the ECAM genes. Several current or potential therapeutics for pathological inflammation work, at least in part, by modulating the activity of transcription factors to inhibit leukocyte adhesion to the endothelium and reduce inflammation in animal models (E. M. Conner, et al., J Pharmacol. Exp. Ther., 282:1615-1622 (1997); J. W. Pierce, et al., J. Immunol., 156:3961-3969 (1996); N. M. Dagia, et al., Am. J Phys., 285:C813-C822 (2003); C. Weber, et al., Circulation, 91:1914-1917 (1995)).
One such group includes methimazole, methimazole derivatives, and tautomeric cyclic thiones (Kohn, L. D., et al., U.S. Pat. No. 6,365,616 Apr. 2, (2002.); Kohn, L. D., et al., U.S. patent application Ser. No. 10/801,986, (2004)). When tested phenylmethimazole (C10), reduced pro-inflammatory (e.g TNF-α)-induced ECAM expression and consequent leukocyte adhesion to endothelial cells (N. M. Dagia, et al., J Immunol, 173:2041-9 (2004)), C10 (i) inhibits monocytic cell adhesion to cytokine inflamed human aortic endothelial cells (HAEC) under in vitro flow conditions that mimic conditions present in vivo; (ii) strongly inhibits cytokine-induced HAEC expression of VCAM-1 at the protein and mRNA level; (iii) has a modest effect on E-selectin expression; and (iv) has very little effect on ICAM-1 expression.
The VCAM-1 promoter contains several cis elements known to play a role in TNF-α induced human VCAM-1 expression: NF-κB, AP-1, SP-1, IRF-1 and GATA. TNF-α stimulation of endothelial cells activates NF-κB (M. J. May, et al., Immunol. Today, 19:80-88 (1998)); however, C10 does not appear to have any effect on NF-κB translocation to the nucleus or binding to the VCAM-1 promoter (N. M. Dagia, et al., J Immunol, 173:2041-9 (2004)). IRF-1 is present at a very low level in resting endothelial cells; however, upon stimulation with TNF-α, IRF-1 is induced, binds to the VCAM-1 promoter, and is necessary for full cytokine-induced transcriptional activation (A. S, Neish, et al., Mol. Cell. Biol., 15:2558-2569 (1995); N. M. Dagia, et al., J Immunol, 173:2041-9 (2004)). C10 inhibits TNF-α induced IRF-1 expression at the protein and mRNA level. While several inhibitors of VCAM-1 are known, very few, if any, have been shown to selectively suppress VCAM-1, to act via IRF-1, and to inhibit monocytic cell adhesion to cytokine inflamed endothelium under fluid shear.
The mechanism of TNF-α induction of IRF-1 in endothelial cells involves Stat1. The IRF-1 promoter region contains two NF-κB binding sites and an activated Stat1-GAS binding sequence (Y. Ohmori, et al., J Biol Chem, 272:14899-907 (1997); H. Ochi, et al., Eur J Immunol, 32:1821-31 (2002)). Although TNF-α-activated NF-κB is directly involved in the activation of IRF-1 gene transcription, NF-κB is, insufficient for full expression and requires Stat1 occupation of the GAS site. Stat1 could be increased by indirect or direct means. Thus, TNF-α could induce IRF-1 promoter activity by its effect on NF-κB, an increase in type I IFN, and the autocrine/paracrine activation of Type I IFN on Stat1 (O. Tliba, et al., J Biol Chem, 278:50615-23 (2003)) Alternatively, TNF-α may directly activate Stat1 since (H. Ochi, et al., Eur J Immunol, 32:1821-31 (2002)), cycloheximide, a protein synthesis inhibitor, does not affect TNF-α induced IRF-1 expression in human umbilical vein endothelial cells (HUVEC), suggesting that TNF-α can induce increased IRF-1 expression without protein synthesis, i.e., without de novo synthesis of IFN.
E. Overexpression of Toll-Life Receptors and Signalling in Autoimmune Inflammatory Disease
Several lines of evidence have emerged in the past several years, which implicate TLRs in inflammatory-autoimmune disorders. For example, constitutive activation of immune cells caused by defective IL-10 signaling results in development of chronic enterocolitis (K. Takeda, et al., Immunity, 10:39-49 (1999)). Introduction of TLR4 deficiency into these mutant mice results in improvement of intestinal inflammation (M. Kobayashi, et al., J Clin Invest, 111:1297-308 (2003)). Development of atherosclerosis observed in apolipoprotein E-deficient mice is rescued by introduction of MyD88 deficiency, implicating the TLR-mediated pathway in the development of atherosclerosis (K. S. Michelsen, et al., Proc Natl Acad Sci USA, 101:10679-84 (2004)). Involvement of the TLR9-MyD88-dependent pathway in the induction of auto-antibodies in SLE and rheumatoid arthritis is described above.
Overexpressed TLR3/TLR4 and TLR3/TLR4Signals in Nonimmune Cells as well as Monocytes, Macrophages, and Dendritic Cells Are Associated with Autoimmune-inflammatory Diseases. Multiple autoimmune inflammatory diseases are now associated with overexpressed TLR3 and TLR4 and or their signals in nonimmune cells, monocytes, macrophages, and dendritic cells. In the case of TLR3/TLR3 signaling, these include Hashimoto's thyroiditis and Type 1 diabetes; in the case of TLR4/TLR4 signaling these include ulcerative colitis, Crohn's, atherosclerosis, and toxic shock. Overexpressed TLR3/4 or TLR3/4 signaling is not limited to these disorders and includes any disease where TLR signaling is activated and increases type I IFNs or cytokine-increased ECAM expression and leukocyte adhesion, e.g., systemic lupus, rheumatoid arthritis, or any autoimmune-inflammatory disease.
Hashimoto's Thyroiditis. It is well recognized that TLR3 on dendritic cells recognize dsRNA, then signal increases in cytokines and recognition molecules important for immune cell interactions. TLR3 mRNA and protein are now recognized to be expressed on thyrocytes and associated with Hashimoto's thyroiditis (N. Harii, et al., Mol Endocrinol, 19:1231-50 (2005)). TLR3 are functional, since incubating thyroid cells with Poly (I:C) causes (i) transcriptional activation of both the NF-κB/Elk1 and IRF-3/IFN-β signal paths, (ii) post transcriptional activation of NF-κB and ERK1/2, and (iii) increased IFN-β mRNA. TLR3 can be overexpressed, along with PKR, major histocompatibility complex (MHC)—I or II, and IRF-1, by transfecting dsRNA into the cells, infection with Influenza A virus, or incubation with IFN-β, but not by incubation with dsRNA or IFN-gamma, or by dsDNA transfection. Methimazole (MMI) and derivatives e.g., phenylmethimazole (C10), significantly prevents overexpression by inhibiting increased transcriptional activation of IRF-3 and ISREs, STAT phosphorylation, but not NF-6β activation. TLR3 can be functionally overexpressed in cultured human thyrocytes by dsRNA transfection or IFN-β treatment. Immunohistochemical studies show TLR3 protein is overexpressed in human thyrocytes surrounded by immune cells in 100% of patients with Hashimoto's thyroiditis examined, but not in normal or Graves' thyrocytes. Without wishing to be bound by theory in any way, it can be concluded that functional TLR3 are present on thyrocytes; TLR3 downstream signals can be overexpressed by pathogen-related stimuli; overexpression can be reversed by C10>>MMI by inhibiting only the IRF-3/IFN-β/STAT arm of the TLR3 signal system; and TLR3 overexpression can induce an innate immune response in thyrocytes which may be important in the pathogenesis of Hashimoto's thyroiditis and in the immune cell infiltrates.
Hashimoto's thyroiditis, the most frequent tissue-specific autoimmune disease in humans, is characterized by infiltration of the thyroid gland by B and T lymphocytes, cellular and humoral autoimmunity, and autoimmune destruction of the thyroid (C. M. Dayan, et al., N Engl J Med, 335:99-107 (1996)). Thyrocytes of patients with Hashimoto's thyroiditis, express ICAM-1, B7-1, essential co-stimulatory molecules important for immune cell interactions, major histocompatibility complex (MHC) class I, interferon (IFN) inducible protein IP-10, a CXCL chemokine that exerts a chemotactic activity on lymphoid cells, and Fas gene, a member of the closely linked group of tumor necrosis factor genes (G. Pesce, et al., J Endocrinol Invest, 25:289-95 (2002); M. A. Garcia-Lopez, et al., J Clin Endocrinol Metab, 86:5008-16 (2001)).
Infectious agents have been implicated in the induction of autoimmune disease (J. Guardiola, et al., Crit Rev Immunol, 13:247-68 (1993); R. Gianani, et al., Proc Natl Acad Sci USA, 93:2257-9 (1996); M. S. Horwitz, et al., Nat Med, 4:781-5 (1998); H. Wekerle, Nat Med, 4:770-1 (1998); C. Benoist, et al., Nature, 394:227-8 (1998)) including thyroiditis (Y. Tomer, et al., Endocr Rev, 14:107-20 (1993)). In the 1990's it was suggested that viral triggering of autoimmunity might result from local infection of tissues, induction of abnormal or increased expression of MHC genes, presentation of self-antigens to immune cells, and bystander activation of T cells (M. S. Horwitz, et al., Nat Med, 4:781-5, (1998); H. Wekerle, Nat Med, 4:770-1, (1998); C. Benoist, et al., Nature, 394:227-8, (1998)).
Endotoxic Shock. A variety of studies have implicated TLR4 in endotoxic shock. For example, C3H/HeJ mice have a point mutation in the Tlr4 gene that results in defects in TLR4 signaling and hypo-responsiveness to challenge with LPS (K. Hoshino, et al., J Immunol, 162:3749-52 (1999)). Recent work (G. Andonegui, et al., J Clin Invest, 111:1011-1020 (2003)) found strong evidence that endothelial TLR4, as opposed to leukocyte TLR4, is a critical player in endotoxic shock. Thus, mice deficient in endothelial TLR4, but not leukocyte TLR4, had significantly attenuated leukocyte sequestration in the lungs subsequent to challenge with LPS.
Cultured murine macrophages, for example RAW 264.7 cells, when treated with LPS display a rapid induction of many genes, which are regulators of the inflammatory response and are considered an in vitro model of changes in endotoxic shock (M. A. Dobrovolskaia, et al., Microbes Infect, 4:903-14 (2002)). LPS stimulated genes in cultured murine macrophages include genes coding for proinflammatory cytokines (IFN-β IL-1β, TNF-α, IL-6, and IL-12), which act on either the macrophages/monocytes themselves or on other target cells to regulate the inflammatory process, which occurs in septic shock. Upon stimulation with LPS, macrophages can also produce CXC chemokines such as IP-10, which serve to further attract immune cells to a site of inflammation (K. M. Kopydlowski, et al., J Immunol, 163:1537-44 (1999)). Macrophages stimulated with LPS can also produce nitric oxide (NO) as a result of expression of the inducible nitric oxide synthase enzyme (iNOS) (C. Bogdan, Nat Immunol, 2:907-16 (2001)). Each of these factors considered to be important in the pathogenesis of septic shock are typically absent or found at extremely low levels in unstimulated macrophages.
Binding of IFN-β to the type I interferon receptor results in phosphorylation of Stat I as a key component for the transduction of a signal to the nucleus to induce expression of iNOS and IP-10 in the mouse macrophage (Y. Ohmori, et al., J Leukoc Biol, 69:598-604 (2001)). Stat1 null animals show an approximately 50% enhanced survival rate when challenged with a lethal dose of LPS (M. Karaghiosoff, et al., Nat Immunol, 4:471-7 (2003)) whereas IFN-β null mice challenged with a lethal LPS dose showed a 100% enhancement of survival (M. Karaghiosoff, et al., Nat Immunol, 4:471-7 (2003)) Therefore, blocking parts of the IFN-β signal pathway is not as effective as blocking the pathway completely.
LPS treatment of macrophage/monocytes increases levels of Interferon Response factor (IRF)-1 (M. A. Dobrovolskaia, et al., Microbes Infect, 4:903-14 (2002)). IRF-1 acts as a transcription factor to directly bind to DNA to enhance transcription of other genes such as iNOS(R. Kamijo, et al., Science, 263:1612-5 (1994)). In macrophages treated with LPS IRF-1 is required for the transcriptional control of the iNOS gene (R. Kamijo, et al., Science, 263:1612-5 (1994)). Several other IRF-1 target genes exist such as the interferon inducible MX gene which codes for the antiviral Mx protein (D. Damino, et al., Curr Opin Cell Biol, 13:454-60 (2001)). The MX promoter has been shown to contain strong IRF-1 binding elements (C. E. Grant, et al., Nucleic Acids Res, 28:4790-9 (2000)).
The proinflammatory cytokines IL-1β, TNF-α, IL-6, and IL-12 can be induced by LPS signaling through TLR4 (M. A. Dobrovolskaia, et al., Microbes Infect, 4:903-14 (2002)) and play a role in endotoxic shock (N. C. Riedemann, et al., J Clin Invest, 112:460-7 (2003)). However, a recent report identified IFN-β as a critical secondary effector, which is induced upon LPS activation of TLR4 signaling and contributes to mortality in a murine septic shock model (M. Karaghiosoff, et al., Nat Immunol, 4:471-7 (2003)).
Inflammatory Bowel Disease (IBD). TLR4 and components of normal gastrointestinal gram-negative bacteria appear to play a key role in the pathogenesis of colitis (C. Fiocchi, Gastroenterology, 115:182-205 (1998); E. Cario, et al., Infect Immun, 68:7010-7 (2000)). The disease is associated with severe inflammation, edema, and leukocyte infiltration in the colonic tissues (C. Fiocchi, Gastroenterology, 115:182-205 (1998); E. Cario, et al., Infect Immun, 68:7010-7 (2000); U. P. Singh, et al., J Immunol, 171:1401-6 (2003); M. B. Grisham, et al., Inflammatory Bowel Disease, 55-64 (1999)). There is increased interferon (IFN) production and secretion and increased levels of cytokines, including TNF-α and IL-1, that up-regulate endothelial cell adhesion molecules (ECAMs), in particular VCAM-1, which are associated with leukocyte adhesion. There are increased chemokine levels such as IP-10 which is known to be colitis related (U. P. Singh, et al., J Immunol, 171:1401-6 (2003)).
Cario et al. (E. Cario, et al., Infect Immun, 68:7010-7 (2000)), reported that TLR4 was upregulated in intestinal epithelial cell lines isolated from patients with IBD. Using the dextran sodium sulfate (DSS)—induced murine model of colitis related to Crohn's and ulcerative colitis, Ortega-Cava et al. (C. F. Ortega-Cava, et al., J Immunol, 170:3977-85 (2003)) found that TLR4 is upregulated in the colon of colitic mice relative to normal mice. Enterocolitis was reported to be significantly improved in TLR4/Stat3-deficient mice, whereas TNF-α/Stat3 deficient mice still had severe enterocolitis, also indicating the importance of TLR4 in mouse models of enterocolitis (M. Kobayashi, et al., J Clin Invest, 111:1297-308 (2003)).
Atherosclerosis and the Vascular Complications of Types 1 and 2 Diabetes, Obesity, and Hypertension: Recent studies have demonstrated the importance of TLR4 in the initiation and progression of atherosclerosis (K. S. Michelsen, et al., Proc Natl Acad Sci USA, 101:10679-84 (2004); G. Pasterkamp, et al., Eur J Clin Invest, 34:328-34 (2004); G. Andonegui, et al., J Clin Invest, 111:1011-1020 (2003)). Thus, mouse knockout studies and studies of human TLR4 polymorphisms have demonstrated that TLR4 plays a role in the initiation and progression of atherosclerosis and vascular disease. Further, (K. S. Michelsen, et al., Proc Natl Acad Sci USA, 101:10679-84 (2004)) mice deficient in endothelial cell TLR4 had a significant reduction in aortic plaque development in atherosclerosis-prone apolipoprotein E-deficient (ApoE−/−) mice and the lack of TLR4 signaling can result in reduced monocyte adhesion to TLR4−/− endothelium.
The model that has emerged is that oxidized LDL, enteroviruses or enterobacteria act as noxious injurious events to increase TLR expression in areas of turbulent blood flow. The increase in the MyD88 pathway, NF-κB, and the cytokine, TNFα, increase VCAM-1 and attract leukocytes. Thus, it is already suggested that it is important to not only block high lipids and or high blood pressure that induce damage at the lesion foci, but also to block pathologic TLR4 induction and signaling causing immune cell attraction and leukocyte adhesion (G. Pasterkamp, et al., Eur J Clin Invest, 34:328-34 (2004)).
Type 1 Diabetes: A recent report has associated overexpressed TLR3 in pancreatic β cells and destructive changes in Type 1 diabetes (L. Wen, et al., J Immunol, 172:3173-80 (2004)). Moreover, the report showed dsRNA could induce insulinitis and type 1 diabetes in animals, consistent with the known animal model wherein coxsacki virus induces Type 1 diabetes in NOD mice. Devendra and Eisenbarth (D. Devendra, et al., Clin Immunol, 111:225-33 (2004)) point out that a wide variety of studies have implicated enteroviruses as a potential agent in the pathogenesis of type 1 diabetes suggesting that the mechanism of viral infection leading to β cell destruction involves the cytokine interferon alpha (IFN-α) [a Type 1 IFN like IFIβ], and hypothesize that activation of TLR by dsRNA and induction of IFN-α, may activate or accelerate immune-mediated beta cell destruction. They conclude (D. Devendra, et al., Clin Immunol, 111:225-33 (2004)) that, “therapeutic agents targeting IFN-α may potentially be beneficial in the prevention of type 1 diabetes and autoimmunity.”
Type I diabetes appears to require a permissive genetic background and an external factor which may be viral. Islet cell antibodies are common in the first months of the disease. They probably arise in part due to β cell injury and represent a primary autoimmune disease. The preeminent metabolic abnormality in Type 1 diabetes is hyperglycemia and glucosuria. Late complications of diabetes are numerous and include increased atherosclerosis with attendant stroke and heart complications, kidney disease and failure, and neuropathy that can be totally debilitating. The link to HLA antigens has been known since 1970. Certain HLA alleles are associated with increased frequency of disease, others with decreased frequency. Increased MHC class I and aberrant MHC class II expression in islet cells has been described (G. F. Bottazzo, et al., N Engl J Med, 313:353-60 (1985); A. K. Foulis, et al., Diabetes, 35:1215-24 (1986)). A definitive link to MHC class I has been made in a genetic animal model of the disease. Thus MHC class I deficiency results in resistance to the development of diabetes in the NOD mouse (D. V. Serreze, et al., Diabetes, 43:505-9 (1994); L. S. Wicker, et al., Diabetes, 43:500-4 (1994)). Combined with recent TLR3 data, and data from Coxsackie virus mouse models, it is hypothesized that infection or environmental induction of Type 1 diabetes occurs in a genetically susceptible mammal, that GAD and anti-islet cell antibodies are abnormal for a prolonged latent phase before total islet cell destruction, and that TLR-induced changes in MHC genes are important in disease expression.
Environmental Inducers of Autoimmune-Inflammatory Disease: The TLR signaling pathway and its pathologic expression in nonimmune cells represents an intriguing link between viral agents and autoimmune-inflammatory disease. For example, multiple viruses have been linked to type 1 diabetes, (e.g., Coxsackie B4 virus) (J. Guardiola, et al., Crit Rev Immunol, 13:247-68 (1993); R. Gianani, et al., Proc Natl Acad Sci USA, 93:2257-9 (1996); M. S. Horwitz, et al., Nat Med, 4:781-5 (1998); H. Wekerle, Nat Med, 4:770-1 (1998); C. Benoist, et al., Nature, 394:227-8 (1998); Y. Tomer, et al., Endocr Rev, 14:107-20 (1993); M. F. Prummel, et al., Thyroid, 13:547-51 (2003); G. S. Cooper, et al., J Rheumatol, 28:2653-6 (2001); M. M. Ward, et al., Arch Intern Med, 152:2082-8 (1992)). The involvement of other “noxious” environmental events is also suspected.
One example of a noxious environmental induction process is tobacco and smoking. Many epidemiologic studies have found a positive association between smoking and autoimmune-inflammatory conditions including rheumatoid arthritis, autoantibodies, Raynaud phenomenon, Goodpasture syndrome, and Graves' disease (I. Roitt, Essential Immunology, 7th ed., 312-346 (1991); S. A. Jimenez, et al., Ann Intern Med, 140:37-50 (2004); C. Nagata, et al., Int J Dermatol, 34:333-7 (1995)). A significant increase in the risk of systemic lupus erythematosus (SLE) has been indicated, as well as rapid development of end-stage renal disease in these patients (G. S. Cooper, et al., J Rheumatol, 28:2653-6 (2001); M. M. Ward, et al., Arch Intern Med, 152:2082-8 (1992)) Smoking is an independent risk factor for diabetes and aggravates the risk of serious disease and premature death (E. B. Rimm, et al., Am J Public Health, 83:211-4 (1993); E. B. Rimm, et al., BMJ, 310:555-9 (1995); N. Kawakami, et al., Am J Epidemiol, 145:103-9 (1997); D. Haire-Joshu, et al., Diabetes Care, 22:1887-98 (1999); J. C. Will, et al., Int J Epidemiol, 30:540-6 (2001)). Results from both cross-sectional and prospective studies show enhanced risk for micro- and macrovascular disease, as well as premature mortality from the combination of smoking and diabetes. On the molecular and cellular levels, a potentially important pathogenic mechanism is the production of chemically altered DNA by reactive elements in cigarette smoke, resulting in the production of autoantibodies specifically against altered DNA (B. H. Hahn, N Engl J Med, 338:1359-68 (1998); J. B. Winfield, et al., J Clin Invest, 59:90-6 (1977)). Additionally, smoking enhances the ability of high glucose levels to affect the walls of the arteries, making them more likely to develop fatty deposits. Smoking enhances a diabetic's chance of having high blood pressure, high levels of lipids such as triglycerides, and lower levels of the protective HDL cholesterol. Cigarette smoking may thus act in concert with other environmental triggers, such as obesity or infectious agents, and can be construed as a major and related environmental factor in the development of diabetes and its complications.
Therefore, it is evident that Hashimoto's thyroiditis may be grouped with insulinitis and Type 1 diabetes, colitis, toxic shock, and atherosclerosis as an autoimmune/inflammatory disease associated with TLR3 or TLR4 overexpression and signaling in nonimmune cells, monocytes, macrophages, and dendritic cells by an induction process involving molecular signatures of environmental pathogens (K. S. Michelsen, et al., Proc Natl Acad Sci USA, 101:10679-84 (2004); G. Pasterkamp, et al., Eur J Clin Invest, 34:328-34 (2004); D. Devendra, et al., Clin Immunol, 111:225-33 (2004); L. Wen, et al., J Immunol, 172:3173-80 (2004); G. Andonegui, et al., J Clin Invest, 111:1011-1020 (2003); C. Fiocchi, Gastroenterology, 115:182-205 (1998); B. Beutler, Nature, 430:257-63 (2004); K. S. Michelsen, et al., J Immunol, 173:5901-7 (2004)). The present invention provides for the use of phenylmethimazoles, methimazole derivatives, and tautomeric cyclic thiones for the treatment of autoimmune/inflammatory diseases associated with TLR3 or TLR4 overexpression and signaling in nonimmune cells as well as monocytes, macrophages, and dendritic cells. It additionally provides for the use of phenylmethimazoles, methimazole derivatives, and tautomeric cyclic thiones for the treatment of autoimmune/inflammatory diseases associated with pathologic activation of TLR signaling involving activation of IRF-3, synthesis of Type 1 IFN, activation of STATs, increased IRF-1 gene expression, and activation of proteins with ISRE elements.